Artificial membranes as model systems to investigate the temperature adaptation of diatoms

在该项目的头两年，我们可以证明，假海链藻和三角褐指藻可以调节它们的脂肪组成，以适应不同的环境条件。在低温条件下培养假单胞菌，导致类囊体中MGDG含量增加，DGDG浓度降低。膜流动性测量表明，MGDG的积累导致膜流动性的增加，即类囊体膜结构的疏松。强光培养显著提高了类囊体膜中SQDG的含量，降低了MGDG的含量。此外，总类囊体脂浓度与叶绿素(Chl)的关系显示出增加的趋势。一般来说，与高等植物相比，硅藻类囊体的类囊体脂/Chl比率明显较高，这表明硅藻细胞膜上的蛋白质拥挤度降低，或者从与Chl结合的蛋白质转移到与色素无关的蛋白质上。这一重要意见将在项目的第三年得到更详细的阐述。膜流动性的进一步测量集中在光保护的叶黄素循环色素二硫杂黄素(DTX)的作用，它在HL条件下被DDX脱环氧酶(DDX)转化。我们发现，在三角星天牛的HL类囊体中，与主要含有DDX的类囊体膜相比，DTX在膜的脂相中含有大量的游离DTX，导致膜硬度增加。在项目的第三年，我们计划对影响硅藻类囊体膜流动性的其他因素进行详细的分析，特别是对硅藻类囊体膜的脂肪酸组成和主导硅藻类囊体膜的阴离子脂类的负电荷。在该项目的头两年，我们开发了利用高效液相色谱与CAD检测器相结合的方法来分析硅藻、脂肪和脂肪酸。我们还开发了一种分离天然硅藻脂肪和脂肪酸的方法，现在将用于构建人造膜系统。除了膜流动性的测量外，这些脂质体还将用于研究脂类和脂肪酸组成的差异对DDX脱环氧化的影响以及捕光络合物(FCP)的结构。在项目的头两年，我们还可以证明FCP的聚集依赖于低pH值和DTX的存在。高浓度的镁离子导致了络合物的进一步聚集。在项目的第三年，还将使用高镁浓度和低pH值来测量膜的流动性和DDX的脱环氧化，以补偿脂质SQDG和PG的负电荷。这将使我们能够确定阴离子脂类的负头基团是否在这些过程中发挥作用。

In the first two years of the project we could show that the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum modulate their lipid composition in adaption to varying environmental conditions. Cultivation of T. pseudonana under lower temperatures induced an increase of the MGDG amount in the thylakoids accompanied by a decreased concentration of DGDG. Measurements of the membrane fluidity indicated that the accumulation of MGDG led to increased membrane fluidity, i.e. loosening of the thylakoid membrane structure. Cultivation under high light conditions strongly increased the percentage of SQDG in the thylakoid membranes and reduced the MGDG content. Furthermore, the total thylakoid lipid concentration, in relation to chlorophyll (Chl), showed an increase. In general, the lipid per Chl ratio is significantly higher in diatom thylakoids compared to higher plants, which indicates either a decreased protein crowding in the diatom membrane or a shift from Chl binding proteins to proteins which are not associated with pigments. This important observation will be addressed in more detail in the third year of the project. Further measurements of the membrane fluidity concentrated on the role of the photoprotective xanthophyll cycle pigment diatoxanthin (Dtx), which is converted from diadinoxanthin (Ddx) by the enzyme Ddx de-epoxidase under HL conditions. We found that in HL thylakoids of P. tricornutum, with a high amount of free Dtx in the lipid phase of the membrane, Dtx leads to increased membrane rigidity in comparison to thylakoids which mainly contain Ddx. In the third year of the project we plan a detailed analysis of the other factors which influence the fluidity of the diatom thylakoid membrane with a special focus on the fatty acid composition and the negative charge of the anionic lipids which dominate the diatom membrane. In the first two years of the project we have developed methods for the analysis of diatom lipids and fatty acids by means of HPLC coupled with a CAD detector. We have also developed a method for the isolation of native diatom lipids and fatty acids which will now be used for the construction of artificial membrane systems. Besides measurements of the membrane fluidity, these liposomes will be used to study the effect of differences in the lipid and fatty acid composition on Ddx de-epoxidation and the structure of light-harvesting complexes (FCPs). During the first two years of the project we could also show that aggregation of the FCPs depends on low pH-values and the presence of Dtx. High concentrations of Mg2+ ions lead to further aggregation of the complexes. In the third year of the project high Mg2+ concentrations and low pH-values will also be used in measurements of the membrane fluidity and Ddx de-epoxidation to compensate the negative charges of the lipids SQDG and PG. This will allow us to determine if the negative head-groups of the anionic lipids play a role in these processes.